[Histonet] glycol methylacrylate GMA enzyme and IHC

[Gayle Callis]

You wrote: 

Hi all,

 

I am going to be exploring some tissue embedding using Glycolmethacrylate

(GMA), as I read that it can preserve enzyme function in some cases and

potentially can be used for IHC. However, information is a bit scant and

can be contradictory at times, to say the least.

 

If anyone has any experience doing any histological or IHC staining on GMA

embedded tissue, or knows of anywhere I could get protocols from or any

papers that are a bit more recent (I'm mostly finding 90s, early 00s),

pleas let me know, I would really appreciate any resources I can find!

 

-- 

Casey Berridge

 

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GMA references from the 80’s and 90’s pretty much remain current for GMA.
Be sure to do a literature search in J Histochem Cytochem (JHC.org) to see
if there are more recent publications.  Google Scholar was not turning up
much for recent years.    

 

We worked with GMA for many years but never for IHC.   There are many
considerations when using GMA both good and bad with emphasis on the
negative side of things from my point of view.   We had success when
studying some single cell protozoa, including Cryptosporidia in a research
setting.   

 

There is one publication in the old Stain Technology, now Biotechnic &
Histochemistry using GMA for successful enzyme staining.  Namba M et al.
Improvement in histochemical demonstration of esterase in glycol
methacrylate tissue sections by cold temperature embedding in glycol
methacrylate. 1983 58(4):207.    

 

Several things about GMA. 

 

Requires a fume hood in order to work with toxic and carcinogenic chemicals.
Glycol methacrylate is sensitizing and several colleagues are so allergic to
fumes after working with this plastic over several years, they can’t be in
the same room where GMA is being worked with.  Double gloving is advisable,
and wearing safety glasses is a must since the sections are small and can
fly into an eye (know of this happening) which is not a good situation.
There should be no skin contact with the plastic, nor breathing the n, n, di
methylaniline, a carcinogen.    Controlling polymerization can be a problem
unless you place embedding molds on top if ice, and cooling the embedding
mixture with ice water.   The polymerization is exothermic, and actually as
blocks polymerize gets uncomfortably hot which may be damaging to enzymes
and sensitive antigens although the heat can be dispersed.   

 

Samples cannot be any thicker than 2 mm, with 1 mm X 1 mm is recommended.
This plastic was first used for liver needle biopsies.    Polymerization for
larger samples is hard to control as is the infiltration by this plastic
hence smaller, thinner samples.   Sectioning is commonly done with glass
knives although tungsten carbide knives work, and I know of one group using
disposable blades with a Leica 2255 model microtome.    More powerful
microtomes i.e. Leica 2650 or equivalent works best.  We had a JB-4
microtome with a special block holder to accommodate the metal
“chucks”/block holders sold by Polysciences.   The metal block holders were
a better heat sink to disperse heat of polymerization.     Sections are
generally no thicker than 1 to 3 µm, and were wonderful when studying single
cell protozoa.  We never used GMA for more routine tissue sections although
it was popular for bone biopsies in clinical labs over the many years.   I
personally found it labor intensive, and expensive for our projects although
the staining results for H&E, PAS-H and some other special stains very nice.


 

Routine stains can be used, including PAS-H, H&E, Massons trichrome with a
modified method, and others.   IHC will not work well, even with JB-4
Immunobed.  GMA, once polymerized, cannot be removed from the section.
Immunobed is probably just a looser matrix than JB-4 and some people have
success.   GMA  plastic is less hydrophobic but still will not allow  large
immunoglobulins to reach antigenic sites.   There has been some success with
IHC but in general, GMA is not the ideal embedding media for immunostaining.
Neil Hand worked with Poly methylmethacrylate for IHC since the plastic can
be completely removed from a thin section, followed by stringent HIER using
a pressure cooker.   PMMA is another world for processing,  sectioning and
staining.  

 

When doing H&E, the staining protocol is different from paraffin section
staining.   If you do an extensive, time intensive search on Histonet, there
are many discussions about GMA staining both for routine and IHC.  

 

You can buy kits, JB-4 and Technovits.   The JB-4 discolors over the years
to a dark tea/brown color making it more difficult to see the tissue while
Technovits remains clear.   When you cut sections, you work with one section
at a time, not a ribbon.   

 

I have a huge file on GMA collected from the early 70’s all the way to
current years.  If you reply to me personally, I can help with references
and  protocols.  
 
In today’s world, if wanting IHC on plastic embedded tissue, I would be
using Neil Hands recommendations and Poly methylmethacrylate (PMMA) plastic
embedding mainly since this plastic is removable.   I also have close
contact with Neil if you need to visit with him.   He is a good plastics
guru and has extensive experience with IHC using PMMA.    Using this plastic
also requires stringent safety precautions for handling the chemicals.    
 
Sorry I leaned towards a negative view of GMA.  
 
Take care
Gayle Callis HTL/HT/MT(ASCP)
GCallis Histology Service, LLC
Bozeman Montana USA